First Report of Pseudomonas tolaasii as the Causal Agent of Bacterial Brown rot on Cauliflower in China

Cauliflower (Brassica oleracea var. botrytis) is extensively cultivated owing to the significant nutritional benefits and substantial economic importance (Song et al. 2019). In January 2024, cauliflower plants showing brown rot symptoms were observed in Xinhua Village, Ansha Town, Changsha City, Hunan Province, China (28°46′N, 113°17′E). Disease incidence was nearly 15% in the planting area (2 ha in size). The infected curds exhibit a water-soaked brown rot, with a foul smell. To identify the causative agent from three curds, infected sections were disinfected with 75% ethanol for 10 seconds, washed twice with distilled water for 15 seconds each time, and then macerated pieces were cut into in the distilled water. The macerates were streaked on nutrient agar medium (Beijing Kuliabo Technology Co., Ltd) and incubated at 28℃ for 48 hours. From the three curds exhibiting identical symptoms, five, six, and four bacterial colonies with similar morphology were isolated, respectively. All isolates displayed smooth, milk-white colonies. One isolate was randomly selected from each curd (HYC24012301, HYC24012302, HYC24012303), as representative for further analysis. The 16S rRNA gene was amplified and sequenced to confirm that three isolated bacteria belong to the genus Pseudomonas (Lalucat et al. 2020). Three housekeeping genes (gyrB, rpoB, rpoD) were amplified with specific primers (Hesse et al. 2018, Roach et al. 2018), respectively. BLAST searches of the sequences revealed 100.0% of 16S rRNA region, gyrB (PP661663-PP661665), rpoB (PP728431-PP728433) and rpoD (PP728428-PP728430) and 100% query coverage to Pseudomonas tolaasii NCPPB 2192T (GenBank accession number PHHD00000000.1). Phylogenetic analysis based on multilocus sequence analysis showed that the three isolates clustered with Pseudomonas tolaasii NCPPB 2192T (Demange et al. 1990). The analysis of the BIOLOG GENIII microplate system indicated that the three strains were negative for sucrose, D-Arabitol, and nitrate reduction, consistent with Pseudomonas tolaasii CFBP 2068T (Milijašević-Marčić et al. 2012), but positive for L-lactic acid and D-glucuronic acid. To assess pathogenicity, cauliflower curds and 30-day-old seedlings (~108•CFUmL-1) were inoculated and incubated at 28℃ and 80% RH in a growth chamber. Negative controls were treated using distilled water. Each isolate was tested in three independent trials, with each treatment comprising both curds and seedlings. Brown rot symptoms were observed in the inoculated curds and leaves three days post-inoculation, consistent with field observations. In contrast, no symptoms were developed in the negative controls. The bacterial strains isolated from symptomatic cauliflower curds and leaves in each group were re-isolated and identified as Pseudomonas tolaasii, thereby fulfilling Koch's postulates. The primary pathogen responsible for mushroom brown blotch is Pseudomonas tolaasii, which has been extensively documented in China (Liu et al. 2022), Spain (González et al. 2009), and Serbia (Svetlana et al. 2012). To our knowledge, this is the first report of Pseudomonas tolaasii causing brown rot in cauliflower. Detailed information about this pathogen on cauliflower is of great significance, as it will help growers implement effective disease management strategies.